Construction of a FRET-based ratiometric fluorescent thiol probe

Article abstract of DOI:10.1039/c0cc03806g

We have rationally constructed a novel FRET-based ratiometric thiol probe suitable for ratiometric imaging in living cells based on the native chemical ligation reaction.

Full text of DOI:10.1039/c0cc03806g

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Construction of a FRET-based ratiometric fluorescent thiol probew
Lingliang Long, Weiying Lin,* Bingbing Chen, Wensha Gao and Lin Yuan
Received 11th September 2010, Accepted 20th October 2010
DOI: 10.1039/c0cc03806g
We have rationally constructed a novel FRET-based ratiometric
thiol probe suitable for ratiometric imaging in living cells based
on the native chemical ligation reaction.
consideration that the emission of Bodipy has a strong overlap
with the absorption of rhodamine. Thus, they are suitable as a
FRET dyad in which Bodipy and rhodamine act as the energy
donor and acceptor, respectively.6 The piperazyl moiety was
chosen as the rigid linker to facilitate the energy transfer
between the Bodipy donor and the rhodamine acceptor. The
distance from the boron atom in the Bodipy dye to the oxygen
atom in the xanthene ring of the rhodamine moiety was
calculated to be 22.88 A (Fig. S2, ESIw). Thus, we expected
that the excited energy of the Bodipy donor could be efficiently
transferred to the rhodamine acceptor in the free NRFTP. To
employ the NCL reaction for the thiol probe design, a
thioester group is required as the potential reaction site for
thiols. Notably, to promote the NCL reaction, we judiciously
selected a thiophenylester moiety as the reaction site of the
probe, as it is a better leaving group than a thioalkylester
group in physiological conditions.5,7 According to the NCL
reaction, we reasoned that when the probe is treated with
Cysteine (Cys), the nucleophilic thiol group of Cys could
attack the electrophilic carbon of the thioester group leading
to the cleavage of the FRET dyad. Since the distance is infinite
after the thiol-induced cleavage of the FRET dyad, the FRET
should be switched off in the presence of thiols. Thus, the
thiol-induced cleavage of the probe may afford the Bodipy
moiety 8 and rhodamine moiety 10. For the formation of the
rhodamine moiety, according to the NCL reaction, a native
amide bond will form between the rhodamine dye and Cys
after the expected trans-thioesterification and intramolecular
rearrangement reactions. At the physiological pH, the resulting
N atom of the amide group may further attack the electro-
philic 9-position carbon of the conjugated xanthene ring to
give the non-fluorescent deconjugated spirolactam compound
10 based on the well-known rhodamine chemistry.
Small-molecular-weight biological thiols, for instance cysteine
(Cys), play a critical role in many biological processes.
However, abnormal levels of Cys are implicated in a variety
of diseases, such as liver damage, skin lesions, and slowed
growth.1 Thus, it is of intense interest to develop sensitive and
selective fluorescent methods2 for the detection of biological
thiols. Some fluorescent probes for thiols have been developed.3
However, a vast majority of them respond to thiols with
optical signal changes only in the fluorescent intensity. The
intensity-based probes can be affected by the variations in the
sample environment. By contrast, ratiometric fluorescent
probes allow the measurement of emission intensities at two
wavelengths, which should provide a built-in correction for
environmental effects.
Forster resonance energy transfer (FRET) is a signaling
¨
mechanism may be exploited for ratiometric fluorescent probe
development. Recently, two research groups constructed
FRET-based thiol probes by linking a fluorescent dye with a
dye quencher.3c,4 However, their FRET-based probes only
exhibited a fluorescence turn-on response to thiols. Thus,
notably, their FRET-based probes are not ratiometric. In this
communication, we describe the construction of the first
FRET-based ratiometric thiol probe based on the native
chemical ligation reaction.
The native chemical ligation (NCL) reaction has been
widely used in peptide synthesis.5 Native chemical ligation of
peptide segments involves reaction between a peptide-a-thioester
and an N-terminal cysteine-peptide. The prominent features of
the NCL reaction include: (1) the reaction is highly chemo-
selective for thiols and devoid of any interference from
other biologically relevant species; (2) the reaction is in vivo
compatible and can proceed inside living cells. It is important
to note that the NCL reaction has not been previously
exploited to design small-molecule fluorescent thiol probes.
In this work, we have judiciously designed a NCL-based
ratiometric fluorescent thiol probe (NRFTP, Fig. 1, Fig. S1
(ESIw)) for ratiometric imaging of thiols in living cells. The
probe is composed of a rhodamine dye, a thioester group, a
piperazyl moiety, and a Bodipy dye. The selection of Bodipy
and rhodamine as the fluorophores is based on the
A versatile new convergent strategy was designed to synthesize
NRFTP in 12 steps. The Bodipy donor building block 2 and
the linker building block 6 were prepared as shown in Schemes
State Key Laboratory of Chemo/Biosensing and Chemometrics,
College of Chemistry and Chemical Engineering, Hunan University,
Changsha, Hunan 410082, P. R. China. E-mail: weiyinglin@hnu.cn;
Fax: +86 731-88821464
w Electronic supplementary information (ESI) available: Experimental
procedures, characterization data for the probe, and some spectra of
the probe. See DOI: 10.1039/c0cc03806g
Fig. 1 Design of ratiometric fluorescent thiol probe NRFTP based on
the NCL reaction.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 893–895 893

View Article Online
Fig. 2 Fluorescence spectra (with excitation at 470 nm) of NRFTP
(1.3 mM) upon addition of increasing concentrations (0–200 mM) of
Cys in 25 mM potassium phosphate buffer (pH 7.4, containing 45%
CH3CN as a co-solvent). The inset shows the changes of fluorescent
intensity ratios at 510 and 590 nm (I510/I590) to increasing concentration
of Cys.
Scheme 1 Synthesis of ratiometric thiol probe NRFTP.
wavelengths is highly desirable for ratiometric probes, as the
sensitivity as well as the dynamic range of ratiometric probes
are controlled by the ratios. The emission ratios have an
excellent linear relationship with the concentrations of Cys
from 1.0 Â 10À7 to 1000 Â 10À7 M (Fig. S7, ESIw), suggesting
that the probe is potentially useful for quantitative determination
of thiol concentrations in a large dynamic range. The detection
limit of the probe was determined to be 8.2 Â 10À8 M (S/N = 3).
A similar ratiometric fluorescence response was noted when
the probe was titrated with other small-molecular-weight
biological thiols, homocysteine or glutathione (Fig. S8, ESIw).
Furthermore, when excited at 550 nm, the fluorescent
intensity of the probe at 590 nm was gradually decreased with
the addition of elevated concentrations of Cys (Fig. S9, ESIw),
consistent with the decreasing of rhodamine absorption at
562 nm (Fig. S6, ESIw). These data suggest that the conjugated
xanthene structure in the rhodamine acceptor was destroyed,
as anticipated. To further confirm the design hypothesis, the
reaction products 8 and 10 of NRFTP with Cys were isolated
and subjected to the standard NMR and mass spectroscopy
characterization (Fig. S12–S15, ESIw), demonstrating that the
ratiometric fluorescence response of the probe to Cys is indeed
due to the NCL reaction.
S1 and S2 (ESIw), respectively. Reaction of amine 6 with
succinimidyl ester 2 in reflux afforded amide 7 (Scheme 1).
The disulfide bond of compound 7 was reduced by NaBH4
at room temperature to give the key intermediate Bodipy
sulfhydryl 8. Rhodamine B was converted into the acid
chloride derivative, which was then coupled with Bodipy
sulfhydryl 8 to provide NRFTP. The reference thioester 9
was prepared by a similar procedure (Scheme S3, ESIw).
The absorption spectrum of NRFTP exhibited the
characteristic absorption of the Bodipy donor with a peak at
500 nm and the typical absorption of the rhodamine acceptor
with a peak at around 562 nm (Fig. S3, ESIw), indicating that
there are little or no electronic interactions between the Bodipy
donor and the rhodamine acceptor in the ground state. When
NRFTP was excited at the Bodipy donor absorption (Fig. S4,
ESIw), the featured emission of the Bodipy donor at around
510 nm was nearly completely quenched when compared with
that of the reference Bodipy donor 8. Concurrently, the
emission of the rhodamine acceptor at around 590 nm
displayed a marked fluorescence enhancement relative to that
of the reference rhodamine acceptor 9 at the same concentration.
This suggests that the excitation energy of the Bodipy donor
is efficiently transferred to the rhodamine acceptor. The
intramolecular energy transfer efficiency from the Bodipy
donor to the rhodamine acceptor in NRFTP was calculated
to be 98.4% (see the ESIw). The efficient intramolecular energy
transfer was collaborated by the excellent correspondence
between the absorption and excitation spectra of the probe
(Fig. S5, ESIw).
The free probe only showed the emission of rhodamine at
590 nm with excitation at 470 nm. However, when NRFTP
was titrated with increasing concentrations of Cys (Fig. 2), the
intensity of the rhodamine emission at 590 nm gradually
decreased with the concomitant growth of a new emission
peak of Bodipy at 510 nm, indicating that the FRET is off. A
well-defined isoemission point was noted at 567 nm. The
changes in the absorption spectra (Fig. S6, ESIw) are in good
agreement with the variation in the emission profile. As shown
in the inset of Fig. 2, the fluorescent intensity ratios at 510 and
590 nm (I510/I590) exhibited a drastic change from 0.09 in the
absence of Cys to 24.82 in the presence of Cys (100 mM), a
275-fold enhancement in the emission ratios. It should be
noted that such a dramatic change of signal ratios at two
The free NRFTP is highly stable in the assay conditions
(Fig. S16, ESIw), and the fluorescence intensity is almost
unchanged with excitation at 470 nm for 30 min (Fig. S17,
ESIw). The time course of the fluorescent intensity ratio
(I510/I590) in the absence or presence of Cys was displayed in
Fig. S18 (ESIw). Upon introduction of Cys, a dramatic
enhancement in the emission intensity ratios was noted, and
the ratios essentially reached the maximum in 20 minutes.
Thus, an assay time of 20 min was selected in the evaluation of
the selectivity and sensitivity of the probe to Cys. The free
probe is stable over a wide pH range of 2.7–10.0, and the
probe can be employed to detect thiols under the physiological
pH value (Fig. S19, ESIw).
To examine the selectivity, NRFTP was treated with various
biologically relevant species including amino acids (Cys, Phe,
Ala, Gly, Glu, Arg, Lys, Tys, Leu, Ser, Val), glucose, metal
ions (K+, Ca2+, Zn2+, Fe3+), a reactive oxygen species
(H2O2), and a reducing agent [nicotinamide adenine dinucleotide
(NADH)]. The probe is highly specific for thiols over other
biologically relevant species (Fig. 3 and Fig. S20 (ESIw)), in
good agreement with the chemoselective nature of the NCL
c
894 Chem. Commun., 2011, 47, 893–895
This journal is The Royal Society of Chemistry 2011

View Article Online
Fig. 4 Confocal fluorescence images of living HeLa cells (excitation
at 488 nm). (a) Bright-field image of HeLa cells incubated with
NRFTP (2 mM) for 30 min; (b) fluorescence image of (a) with emission
at 515 Æ 10 nm; (c) fluorescence image of (a) with emission at
610 Æ 10 nm; (d) overlay of the images of (a), (b), and (c).
(e) Bright-field image of HeLa cells pre-treated with 1 mM N-ethyl-
maleimide for 30 min, and then incubated with the probe (2 mM) for
30 min; (f) fluorescence image of (e) with emission at 515 Æ 10 nm;
(g) fluorescence image of (e) with emission at 610 Æ 10 nm; (h) overlay
of the images of (e), (f), and (g).
Fig. 3 Ratiometric response (I510/I590) of NRFTP (1.3 mM) to
100 mM of various species: (1) KCl; (2) CaCl2; (3) ZnCl2; (4) FeCl3;
(5) Cys; (6) glucose; (7) NaDH; (8) H2O2; (9) Phe; (10) Ala; (11) Gly;
(12) Glu; (13) Arg; (14) Lys; (15) Tys; (16) Leu; (17) Ser; (18) Val.
lex = 470 nm.
reaction. In addition, the visual response of the probe
to various species indicates that the probe can be used
conveniently for thiol detection by simple visual inspection
(Fig. S21, ESIw).
sensitivity, a large ratio signal variation, and a large linear
dynamic range. The probe has been applied for ratiometric
detection of thiols in biological fluids. Importantly, the
FRET-based ratiometric imaging of thiols in living cells has
been demonstrated by employing NRFTP. Thus, we expect
that NRFTP will be a useful molecular tool for diverse
biological applications including the determination of thiol
levels in biological fluids, fluorescence labelling of proteins,
and the assaying of enzymes with a thiol as a product for
enzyme–inhibitor screening. Furthermore, the NCL-based
thiol probe design concept should be widely applicable for
construction of ratiometric thiol probes.
Probe NRFTP was then applied for thiol detection in real
biological samples. Aliquots of reduced serum sample were
added to a solution of NRFTP (1.3 mM) (see the ESIw). As
exhibited in Fig. S22 (ESIw), the increase in the amount
of newborn-calf serum elicited a linear enhancement in the
emission ratios (I510/I590), suggesting that the probe is capable
of ratiometric sensing of thiols in the plasma sample. For
quantitative measurement, a standard addition method with
Cys as the standard was used to estimate the unknown
concentration of total thiols in a human urine sample from a
healthy volunteer. The total content of thiols in the urine
sample was analyzed to be 40 Æ 3 mM, which is well within the
reported thiol concentration range for the urine samples from
the healthy individuals.8
This research was supported by NSFC (20872032,
20972044), NCET (08-0175), and the Key Project of Chinese
Ministry of Education (108167).
The probe has relatively low toxicity to the HeLa cells
(Fig. S23, ESIw). The suitable amphipathicity and low toxicity
of probe NRFTP may render it applicable for ratiometric
imaging in living cells. To examine this possibility, the living
Hela cells were treated with NRFTP. The dual-channel
fluorescence images recorded at 515 Æ 10 nm and 610 nm Æ
10 nm with excitation at 488 nm were shown in Fig. 4.
Incubation of living HeLa cells with NRFTP (2 mM) for 30
min provided a strong green fluorescence in the Bodipy
emission window (Fig. 4b) and almost no red fluorescence in
the rhodamine emission window (Fig. 4c). By contrast, in a
control experiment, the Hela cells were pre-treated with
N-ethylmaleimide (1 mM, as a thiol-reactive reagent) for
30 min, and further incubated with the probe (2 mM) for
30 min, giving intense red fluorescence (Fig. 4g) but essentially
no green fluorescence (Fig. 4f), in good agreement with the
emission profile of the free probe. The ratiometric imaging
data are shown in Fig. S24 (ESIw). Thus, these results establish
that NRFTP is cell membrane permeable and able to display
FRET-based ratiometric fluorescence response to intracellular
thiols.
Notes and references
1 S. Shahrokhian, Anal. Chem., 2001, 73, 5972–5978.
2 (a) A. P. Demchenko, Introduction to Fluorescence Sensing,
Springer, 2008; (b) J. R. Lakowicz, Topics in Fluorescence
Spectroscopy, Probe Design and Chemical Sensing, Kluwer
Academic Publishers, New York, 2002, vol. 4.
3 (a) X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010,
39, 2120; (b) N. Shao, J. Jin, H. Wang, J. Zheng, R. Yang, W. Chan
and Z. Abliz, J. Am. Chem. Soc., 2010, 132, 725; (c) H. Shiu,
H. Chong, Y. Leung, M. Wong and C. Che, Chem.–Eur. J., 2010,
16, 3308; (d) J. H. Lee, C. S. Lim, Y. S. Tian, J. H. Han and
B. R. Cho, J. Am. Chem. Soc., 2010, 132, 1216; (e) S. Sreejith,
K. P. Divya and A. Ajayaghosh, Angew. Chem., Int. Ed., 2008, 47,
7883; (f) B. Zhu, X. Zhang, Y. Li, P. Wang, H. Zhang and
X. Zhuang, Chem. Commun., 2010, 46, 5710–5712; (g) X. Li,
S. Qian, Q. He, B. Yang, J. Li and Y. Hu, Org. Biomol. Chem.,
2010, 8, 3627.
4 P. K. Pullela, T. Chiku, M. J. Carvan III and D. S. Sem, Anal.
Biochem., 2006, 352, 265.
5 P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. H. Kent,
Science, 1994, 266, 776–779.
6 X. Zhang, Y. Xiao and X. Qian, Angew. Chem., Int. Ed., 2008, 47,
8025–8029.
7 D. Bang, B. L. Pentelute, Z. P. Gates and S. B. Kent, Org. Lett.,
2006, 8, 1049.
8 (a) T. Inoue and J. R. Kirchhoff, Anal. Chem., 2002, 74, 1349;
(b) M. P. Brigham, W. H. Stein and S. Moore, J. Clin. Invest., 1960,
39, 1633.
In summary, the FRET-based ratiometric thiol probe
NRFTP was constructed based on the NCL reaction. The
favorable features of the probe include high stability and
functioning well at physiological pH, high selectivity, high
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 893–895 895